Chemiluminescent enzyme immune assays

Initial blood sample was taken from the antecubital vein after 30 min of rest. Blood samples were used to measure blood glucose, lactate, serum insulin, and free fatty acid (FFA), creatine kinase (CK), myoglobin (Mb) and plasma IL-6 concentrations. Serum and plasma samples were obtained by centrifuging at 3000 rpm for 10 min at 4 °C. The plasma and serum samples were stored at −60 °C until analysis. Blood glucose and lactate concentrations were measured using an automatic glucose analyzer (Free style, Nipro Corporation, Osaka, Japan) and lactate analyzer (Lactate pro, Arkray Inc, Kyoto, Japan), respectively. Serum insulin and FFA concentrations were measured using chemiluminescent enzyme immune assays (Fujirebio Inc., Tokyo, Japan) at a clinical laboratory (SRL Inc., Japan). Serum CK and Mb concentrations were also measured at the SRL Clinical Laboratory in Tokyo, Japan. The plasma IL-6 concentration was assayed with an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). The intra-assay coefficients of variation for each measurement were as follows: 3.1 % for insulin, 1.3 % for FFA, 2.8 % for CK, 2.4 % for Mb and 6.6 % for IL-6.

Blood glucose, lactate, serum insulin, free fatty acid (FFA), and glycerol were measured from the whole-blood and serum samples obtained. Serum samples were obtained from blood by centrifugation for 10 min, and were stored at −80°C until analysis. Blood glucose and lactate concentrations were measured immediately after blood collection. Concentrations of blood glucose and lactate were determined using an automatic glucose analyzer (Free Style; Nipro Corporation, Osaka, Japan) and a lactate analyzer (Lactate Pro 2; Arkray Inc. Kyoto, Japan), respectively. The samples for glucose concentrations were analyzed in duplicate. The intraclass coefficient for duplicate measurements in the analysis was 0.99. Serum insulin and FFA concentrations were measured using chemiluminescent enzyme immune assays (Fujirebio Inc., Tokyo, Japan) at a clinical laboratory (SRL Inc., Japan). Serum glycerol concentrations were measured in duplicate using an enzyme-linked immunosorbent assay (Cayman Chemical Company, Ann Arbor, MI, USA). The intra-assay CVs were as follows: 3.3% for serum insulin, 2.2% for serum FFA, and 1.2% for serum glycerol measurements.

Blood glucose, lactate, serum insulin, free fatty acid (FFA), glycerol, and cortisol concentrations were measured using whole-blood or serum samples. Serum samples were obtained by centrifugation for 10 min, and were stored at –80°C until analysis. The blood glucose and lactate concentrations were measured immediately after blood collection. Blood glucose and lactate concentrations were determined using an automated glucose analyzer (Free Style; Nipro Corporation, Osaka, Japan) and lactate analyzer (Lactate Pro 2; Arkray Inc., Kyoto, Japan), respectively. The glucose concentrations were analyzed in duplicate. The intraclass coefficient for duplicate measurements was 0.99. Serum insulin and FFA concentrations were measured by chemiluminescent enzyme immune assays (Fujirebio Inc., Tokyo, Japan) at a clinical laboratory (SRL Inc., Tokyo, Japan). Serum glycerol concentrations were measured in duplicate by enzyme-linked immunosorbent assay (Cayman Chemical Company, Ann Arbor, MI). Serum cortisol concentrations were measured by radioimmunoassay (RIA) using commercially available kits (Immunotech, Marseille, France). The intraassay coefficients of variation (CVs) were 1.1% for serum insulin, 2.2% for serum FFA, 3.3% for serum glycerol, and 4.4% for serum cortisol measurements.